Note: Descriptions are shown in the official language in which they were submitted.
2153733
- 1 -
IMMUNOGT~,NIC OLIGOSACCHARIDE COMPOSITIONS
Field of the Invention:
This application relates to immnnogenic oligosaccharide compositions and
methods of making and using them. In particular, the compositions comprise
oligosaccharides covalently coupled to carrier protein, wherein the resultant
conjugate elicits an immlln~protective response.
Refe~ellces:
1 0 The following references are cited in the application at the relevant
portion of the application.
Anderson, P., Infect. Tmmnn. 39:233, 1983
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2153733
Bolan, G., Broome, C. V., Fracklam, R.R." Pitkaytis, B.D., Fraser, D.
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Fattom, A., Lue, C., Szu, S. C., Mestecky, J., Scllirrlll~, G., Brylar,
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21~7~3
Goebel, W. F. and Avery, O. T., J. Exp. Med. 50: 521, 1929.
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Pahor, A., Clin. Exp. Immunol. 93:157, 1993.
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Herzenberg, C. Blackwell and L. A. Herzenberg, eds., Blackwell, Oxford,
1986.
Heidelberger, M. and Avery, O. T., J. Exp. Med. 38:73,1923.
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1 5 Weibel, R. E., and Woodhour, A. F., Rev. Infect. Dis. 3 (suppl):S31, 1981.Jennings, H. J., and Lugowski, C., Tmml~nogenic polysaccharide-protein
conjugates, C~n~ n Patent No. 1,181,344, 1985.
Jennings, H. J., Roy, R., and G~mi~n, A. J., Modified meningococcal
group b polysaccharide for conjugate vaccine, C~n~ n Patent No. 1,261,320,
1989.
Jones, J.K.N. and Perry, M.B., J. Am. Chem. Soc. 79:2787, 1957.
Kenne, L. and Lindberg, B., Bacterial polysaccharides in "The
polysaccharides - Vol 2", G. O. Aspinall, Ed., ~c~lernic Press, New York,
1983.
Lee, C-J., Banks, S. D. and Li, J. P., Crit. Rev. Microbio. 18:89, 1991.
Lees, A., Finkelman, F., Inman, J.K., Witherspoon, K., Johnson, P.,
Kennedy, J. and Mond, J.J., Vaccine 12:1160, 1994.
Lock, R. A., T-T~n~m~n, D., and Paton, J. C., Microbial Pathogen.
12:137, 1992.
2I5373~
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Madore, D. V., Jackson, C. L., Phipps, D. C., Penridge Pediatric
Association, Popejoy, L.A., Eby, R., and Smith, D. H., Pediatric, 85: 331,
1990.
Malcolm, A. J., Messner, P., Sleytr, U. B., Smith, R. H., and Unger, F.
5 M., Crystalline bacterial cell surface layers (S-layers) as combined
carrier/adju~ for conjugate vaccines, in "Immobilized Macromolecules:
Application Potentials," U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds,
Springer-Verlag, London, 1993a.
Malcolm, A. J., Best, M. W., Szarka, R. J., Mosleh, Z., Unger, F. M.,
1 0 Messner, P. and Sleytr, U. B., Surface layers of Racill~/~ alvei as a carrier for a
Streptococcus pneumoniae conjugate vaccine in "Adances in Bacterial
Paracrystalline Surface Layers," T. J. Beveridge and S. F. Koval, eds., Plenum
Press, New York, 1993b.
Mandell, G. L., "Principles and Practice of Infectious Diseases, "
1 5 Churchill Livingston, New York, 1990.
Marburg, S., Jorn, D., Tolman, R. L., Arison, B., McCauley, J.,
Kniskern, P. J., Hagopian, A., and Vella, P.O., J. Am. Chem. Soc. 108:5282,
1986.
Marburg, S., Tolman, R. L., and Kniskern, P. J., Covalently-modified
20 polyanionic bacterial polysaccharides, stable covalent conjugates of such
polysaccharides and immllnt)genic proteins with bigeneric spacers, and methods
of prel,a~ g such polysaccharides and conjugates and of co"r" ",i~-g covalency,
U.S. Patent No. 4,695,624, 1987.
Marburg, S., Kniskern, P. J., and Tolman, R. L., Covalently-modified
25 bacterial polysaccharides, stable covalent conjugates of such polysaccharides and
immlmogenic proteins with bigeneric spacers and methods of prepalillg such
polysaccharides and conjugates and of collrll ~ g covalency, U. S. Patent No.
4,882,317, 1989.
Mufson, M. A., Hughey, D., and Lydick, E., J. Infect. Dis. 151:749,
30 1985.
2153733
Mufson, M. A., Krause, H. E., Schiffm~n, G., and Hughey, D. E.,
Am. J. Med. Sci. 293: 279, 1987.
Nielsen, S. V., and Henrichsen, J., Scand. J. Infect. Dis. 25:165, 1993.
Paton, J. C., Lock, R. A., Lees, C-J., Li, J. P., Berry, A. M., Mitchell,
5 T. J., Andrew, P. W., T-T~n~m~n, D., and Boulnois, G. J., Infect. Tmmlln.
59:2297, 1991.
Peeters, C.C.A.M., Tenbergen-Meekes, A-M., Poolman, J. T., Berutett,
M., Zegers, B. J. M. and Rijkers, G. T., Infect. Tmmlm. 59: 3504, 1991.
Penney, C.L., Michon, F., and Jennings, H.J., Improved Vaccine
1 0 Compositions, WO 92/04951, 1992.
Pe~ lu~lel, R. M., Hansburg, D., Briles, D. E., Nicolotti, R. A., and
Davie, J. M., J. Tmmllnt)l. 121:566, 1978.
Porro, M., Oligosaccharide Conjugate Vaccines, C~n~ n Patent No. 2
052 323, 1992.
1 5 Porro, M., and Costantino, P., Glycoploteilleic conjugates having
trivalent immllnngenic activity, U. S. Patent No. 4,711,779, 1987.
Porro, M., Oligosaccharide conjugate vaccines, U.S. Patent Application
No. 07/590,649, 1990.
Saunders, L.A.M., Rijkers, G. T., Kuis, W., Tenbergen-Meekes, A. J.,
20 de Graff-Meeker, B. R., Hiemstra, I. and Zegers, B. J. M., J. Allergy Clin.
Immunol. 91: 110, 1993
Schidt, R. A., Boyd, J. F., McCracken, J. D., Scllirr",~n, G., and
Giolma, J. P., Med. Pediatr. Oncol. 11:305, 1983.
Schneerson, R., Barrera, O., Sutton, A., and Robins, J. B., J. Exp. Med.
25 152:361,1980.
Schneerson, R., Robbins, J. B., Chu, C., Sutton, A., Vann, W.,
Vickers, J. C., London, W. T., Curfman, B., and Hardegree, M. C.,Infect.
Tmml-n. 45:582, 1984.
- 6 - 21~37~3
Schneerson, R., Robbins, J. B., Parke, J. C., Bell, C., Schlesselman, J.
J., Sutton, A., Wang, Z., Schiffm~n, G., Karpas, A., and Shiloach, J., Infect.
Tmmlln. 52:519, 1986.
Schneerson, R., Levi, L., Robbins, J. B., Bryla, D. M., Scllirr~ n, G,
5 and Lagergard, T., Infect. and Tl~llllllnily 60:3528, 1992.
Seid, R. C., Jr., Boykins, R.A., Liu, D. F., Kibrough, K. W., Hsieh,
C.L., Eby, R., Glycoconj. J. 6: 489, 1989.
Sell, S. H., Wright, P. F., Vaughn, W. K., Thompson, J., and
ScllirrlllAll~ G., Rev. Infect. Dis. 3 (suppl):S97, 1981.
1 0 Shapiro, E.D., and Clemens, J. D., Ann. Intern. Med. 101 :325, 1984.
Shapiro, E. D., N. Engl. J. Med. 316:1272, 1987.
Shapiro, E. D., Pneumococcal vaccine, In: "Vaccines and
Tmmllnotherapy," S. J. Fryz Jr., ed., Pergamon Press, New York, 1991.
Siber, G. R., Weitzman, S. A., Aisenberg, A. C., Weinstein, H. J., and
1 5 scllirrlllAll~ G., N. Engl. J. Med. 299:442, 1978.
Simberkoff, M. S., Cross, A. P., Al-Ibrahim, M., Baltch, A. L.,
Geiseler, P. J., Nadler, J., Richmond, A. S., Smith, R. P., Scl-irrll~ , G., andShepard, D. S., N. Engl. J. Med. 315:1318, 1986.
Simberkoff, M. S., Pneumococcal vaccine in adults, In: "T~ lion,"
20 M. A. Sande, and R. K. Root, ed., Churchill Livingstone, New York, 1989.
Sims, R. V., Steinman, W. C., McConville, J. H., King, L. K., Zwick,
W. C., and Schwartz, J. C., 1988, Ann. Intern. Med., 108:653., 1988.
Slack, J., Der-Balian, G. P., Nahm, M. and Davie, J. M., J. Exp. Med.
151:853-1980.
Sorensen, U. B.S., J. Clin. Micro. 31: 2097, 1993.
Sloyer, J. L., Jr., Ploussard, J. H., and Howie, V. M., 1981, Rev.
Infect. Dis. 3 (suppl):Sl, 1981.
Stein, K. E., J. Inf. Dis. 165: 549, 1992.
Stein, K. E., Int. J. Tech. Assess, Health Care, 10: 167, 1994.
2153733
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Stein, K. E., Zopf, D. A., Johnson, B. M., Miller, C. B. and Paul, W.
E., J. Tmmllnol. 128: 1350, 1982.
Stein, K. E., Zopf, D. A. and Miller, C. B., J. Exp. Med., 157: 657,
1983.
Steinhoff, M.C., Edwards, K., Keyserling, H., Thoms, M. L., Johnson,
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1 0 The disclosure of the above publications, patents and patent applications
are herein incorporated by l~r~lellce in their entirety to the same extent as if the
language of each individual publication, patent and patent application were
specifically and individually included herein.
1 5 B~l~k~E~round of thP InvPntion:
Tl..-..~..-~ Responses to Poly~ h~rides
Heidelberger and Avery (1923) demonstrated that the type specific
antigens of pneumococci are polysaccharides. Bacterial capsular polysaccharides
20 are cell surface antigens composed of identical repeat units which form extended
saccharide chains. Polysaccharide structures are present on pathogenic bacteria
and have been identified on Escherichia coli, Neisseria meningitidis,
Haemophilus influenzae, Group A and Group B Streptococcus, Streptococcus
pneumoniae and other species. (Kenne and Lindberg 1983).
Specific blood group d~te- lllin~ i and "tumor-associated" antigens are
examples of ~ n cell surface carbohydrates. Oncogenically l~ ro~ ed
cells often display surface carbohydrates distinctly dirr~lclll from those of non-
transformed cells. These glycans consist of only a few monosaccharides
30 (Hakomori and K~nn~gi 1986). The glycan structures by themselves are usually
`_ 2153733
not antigenic, but constitute haptens in conjunction with protein or glycoploteimatrices.
A general feature of saccharide antigens is their inability to elicit
5 significant levels of IgG antibody classes (IgG isotypes) or memory responses,they are considered thymus-independent (TI) antigens. Conjunction of
polysaccharide antigens or of immlmnlogically inert carbohydrate haptens to
thymus dependent (TD) antigens such as pfoleills enhAnres their immlmogenicity.
The protein stimlllAtes carrier-specific T-helper cells which play a role in the1 0 induction of anti-carbohydrate antibody synthesis (Bixler and Pillai 1989).
Much of our current knowledge of TI and TD responses comes from
studies of pertinent mouse models (Stein et al., 1983; Stein, 1992; Stein, 1994).
TI antigens generally elicit low affinity antibodies of restricted class and do not
1 5 produce immlmnlogic memory. Adjuvants have little effect on response to TI
antigens. In contrast, TD antigens elicit heterogeneous and high affinity
antibodies with i~ lni~;ltion and produce immunologic memory. Adjuvallls
enhance response to TD antigens. Secondary responses to TD antigens shows an
increase in the IgG to IgM ratio, while for TI antigens the secondary response
20 IgG to IgM ratio is one-to-one, similar to that of a plilllaly response (Stein et al.,
1982; Stein, 1992 and 1994). In mice and hllmAn~, TD antigens elicit
predo~ A ~Illy IgGl isotypes, with some amounts of IgG2 and IgG3 isotypes. TI
responses to polysaccharides are restricted to IgG3 of the IgG isotypes
(Pelllllullel et al., 1978; Slack et al., 1980).
Current Pneumococcal Vaccine
Pneumococci are ~;ull~lllly divided into 84 serotypes based on their
capsular polysaccharides. Although there is some variability of commonly
occurring serotypes with geographic location, generally serotypes 1, 3, 4, 7, 8
30 and 12 are more prevalent in the adult population. Serotypes 1, 3, 4, 6, 9, 14,
21S37~3
-
18, 19 and 23 often cause pneumonia in children (Mandell, 1990; Connelly and
Starke, 1991; Lee et al., 1991; Sorensen, 1993; Nielsen and Henricksen, 1993).
At present, the most widely used anti-pneumococcal vaccine is composed
5 of purified capsular polysaccharides from 23 strains of pneumococci
(Pneumovax~23, Merck Sharp & Dohme). The pneumococcal capsular types
included in Pneumovax~23 are 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, lOA, 1 lA,
12F, 14, 15B, 17F, 18C, l9F, l9A, 20, 22F, 23F, 33F (Danish nomenclature).
These serotypes are said to be responsible for 90 percent of serious
1 0 pneumococcal disease in the world.
Some controversy exists in the literature over the efficacy of the
Pneumovax~23 vaccine (Borgano et al.,1978; Broome et al., 1980; Sloyer et al.,
1981,; Shapiro and Clemens, 1984; Bolan et al., 1986; Simberkoff et al., 1986;
1 5 Forester et al., 1987; Shapiro, 1987; Sims et al., 1988; Simberkoff, 1989;
Shapiro, 1991). The pneumococcal vaccine is effective for induction of an
antibody response in healthy young adults (Hilleman et al., 1981; Mufson et
al., 1985; Bruyan and van Furth, 1991). These antibodies have been shown to
have in vitro opsonic activity (Chudwin et al., 1985). However, there is marked
20 variability in the hllellsily of the response and in the persistence of antibody titers
to the different serotypes (Hilleman et al., 1981; Mufson et al., 1987).
Children under 2 years of age are the group at highest risk of systemic
disease, otitis media and acute lower re~pilatoly infection caused by
25 pneumococci, but they do not respond to this vaccine (Sell et al., 1981;
Hazelwood et al., 1993; Saunders et al., 1993). Furthermore, elderly and
immunosuppressed patients have impaired or varied responses to Pneumovax~23
(Siber et al., 1978; Schildt et al., 1983; Forester et al., 1987; Simberkoff, 1989;
Shapiro, 1991). These population groups do not respond well to the thymus
30 independent polysaccharide antigens of this vaccine. Typical of thymus
_ 21S37~
- 10-
independent antigens, antibody class switching from an IgM to IgG isotype is notusually observed nor is an ~n~mn~stic response to a booster i~ ion
(Borgano et al., 1978).
Recent occurrences of antibiotic resistant strains of bacteria stresses the
need to develop efficacious vaccines for the prevention of childhood infection.
Clearly, new vaccines against pneumococci are n~e~le~l especially for high risk
groups and children.
10 Conjugate Vaccines
Avery and Goebel were the first to prepare vaccines against bacterial
infections (Avery and Goebel 1929; Goebel and Avery 1929). More recently,
several protein carrier conjugates have been developed which elicit thymus
dependent responses to a variety of bacterial polysaccharides. To date, the
1 5 development of conjugate vaccines to Hemophilus influenzae type b (Hib) has
received the most attention. Schneerson et al. (1980) have covalently coupled
Hib polysaccharides (polyribitol-phosphate) to diphtheria toxoid. This group hasalso developed a Hib vaccine by derivatizing the polysaccharide with an adipic
acid dihydrazide spacer and coupling this material to tetanus toxoid with
20 carbodiimide (Schneerson et al., 1986). A similar procedure was used to
produce conjugates cont~ining diphtheria toxoid as the carrier (Gordon, 1986 and1987). A bifunctional spacer was utilized to couple the outer membrane protein
of group B Neisseria meningitidis to Hib polysaccharides (Marburg et al., 1986,
1987 and 1989). Finally, Anderson (1983 and 1987) has produced a conjugate
25 vaccine using Hib oligosaccharides coupled by reductive amination to a nontoxic,
cross-reactive mutant diphtheria toxin CRM~97.
Reports in the literature differ on the efficacy of these vaccines, and many
studies are still in progress. However, oligosaccharide conjugates (Anderson et
30 al., 1985a, 1985b, 1986, 1989; Seid et al., 1989; Madore et al., 1990; Eby et
21 $3 7~3
- 11 -
al., 1994) and polysaccharide conjugates (Barra et al., 1993) are reported to beimmlmogenic in infants and elicit a thymus dependent response. Hapten loading
is a key factor for conjugate immunogenicity (Anderson et al., 1989; Eby et al.,1994).
Other conjugate vaccines have been developed by Jennings et al. (1985
and 1989), who utilized periodate activation to couple polysaccharides of
Neisseria meningitidis to tetanus or diphtheria toxoid carriers. Porro (1987)
defined methods to couple esterified N. meningitidis oligosaccharides to carrier1 0 proteins. Conjugate vaccines cont~ining polysaccharides of Pseudomonas
aeruginosa coupled by the periodate procedure to detoxified protein from the
same organism (Tsay and Collins, 1987) have been developed. Cryz and Furer
(1988) used adipic acid dihydrazide as a spacer arm to produce conjugate
vaccines against P. aeruginosa.
Polysaccharides of specific serotypes of S. pneumoniae have also been
coupled to classical carrier proteins such as tetanus or diphtheria toxoids
(Schneerson et al., 1984; Fattom et al., 1988 and 1990; Schneerson et al.,
1992), to N. meningitidis membrane protein (Marburg et al., 1987; Giebink et
20 al., 1993) and to a pneumolysin mutant carrier (Paton et al., 1991; Lock et al.,
1992; Lee et al., 1994). Technology for coupling S. pneumoniae
oligosaccharides to CRMl97 protein has been developed (Porro, 1990). These
conjugate vaccines have variable or as yet und~te~ ed immlmopotentiation
properties. Reproducibility of these coupling technologies with the maintenance
25 of immllnogenic epitopes is currently the greatest problem in developing effective
S. pneumoniae glyco-conjugate vaccines. The optimal immllnogenic
oligosaccharide size appears to vary dependent on the serotype, in-lic~ting a
conformational aspect of certain immunogenic epitopes (Eby et al., 1994;
Steinhoff et al., 1994).
- 12- 2153733
Vaccines to DTP, tuberculosis, polio, measles, hepatitis, Hib and
pneumonia which induce long lasting protection are n~e~e(l. In order to induce
protection in infants to S. pneumoniae, a multi-hapten protein conjugate
cont~ining a high level of oligosaccharides of optimal immllnogenic size for each
5 serotype is desired.
Various researchers have proposed enhancement of the immllnogenicity
of conjugate vaccines by adjuvant a(lmini.~tration. Al~ lill.l.ll salt, which isapproved for human use, is an example. Carbohydrate moieties, such as beta
10 glucan particles and low molecular weight dextran, have also been reported topossess adjuvant activity. Adjuvax (Alpha-Beta Technology) is an adjuvant
composition cont~ining beta glucan particles. Lees et al. (1994) have reported
the use of low molecular weight dextran constructs as adju~ . Penney et al.
(1992) have reported a long chain alkyl compound with immunological activity.
Brief Description of the D~awi.~:
Figure 1 illustrates the repeat unit structures of the polysaccharides used
in the Examples of the invention.
Figure 2 shows the separation profile of Streptococcus pneumoniae
serotype 8 capsular polysaccharides through a BioGel P-10 column after acid
hydrolysis (0.5 M trifluoroacetic acid, 100C, 20 minutes) resulting in
discernible oligosaccharides of one to eight repeat units.
Figure 3 shows the relative size of the repeat units in peaks 1, 2, 3 and 4
of hydrolyzed Streptococcus pneumoniae serotype 8 capsular polysaccharides, as
measured by HPLC analysis.
21 S3 733
- 13 -
Figure 4 shows the HPLC retention times of the glucose, M-3
maltotriose, M-7 maltoheptose, and M-10 malto-oligosaccharide standards used
to determine the relative size of various oligosaccharide repeat units.
Figure 5 is an example of the retention times of ribitol, rhamnose,
galactose, fucose and mannose monosaccharide standards used to d~Le~ e
carbohydrate content of the hydrolysed repeat unit.
Figure 6 shows the separation profile of S. pneumoniae serotype 6B
polysaccharide hydrofluoric acid hydrolysates passed over a P-10 BioGel
column.
Figure 7 shows the separation profile of S. pneumoniae serotype 6B
polysaccharide TFA hydrolysates passed over a P-60 BioGel column.
1 5
Figure 8 shows the separation profile of S. pneumoniae serotype 14
polysaccharide TFA hydrolysates passed over a P-30 BioGel column.
Figure 9 shows a separation profile of S. pneumoniae serotype l9F
polysaccharide acetic acid hydrolysates acetic acid passed over a P-10 BioGel
column.
Figure 10 shows the separation profile of S. pneumoniae serotype 23F
polysaccharide TFA hydrolysates passed over a P-10 BioGel column.
Figure 11 shows the separation profile of S. pneumoniae serotype 8
polysaccharide cleaved by cellulase passed over a P-10 Bio Gel column.
21~3733
- 14 -
Figure 12 shows the separation profile of pneumococcal C-substance
polysaccharide hydrofluoric acid hydrolysates passed over a P-10 Bio Gel
column.
Figure 13 shows the inhibition ELISA results using a mouse antiserum to
Streptococcus pneumoniae serotype 8 oligosaccharide protein carrier conjugate.
Figure 14 illustrates the acidification of oligosaccharides for carbodiimide
coupling.
Figure 15 shows the separation of reduced and periodate fractions of a
polysaccharide (23 valent polysaccharide vaccine-Pneumovax~ 23, Merck, Sharp
and Dohme).
Figure 16 demon~lldt~s separation of reduced and periodate fractions of
oligosaccharides of serotype 6B of Streptococcus pneumoniae.
Figure 17 demonstrates separation of reduced and periodate fractions of
oligosaccharides of serotype l9F of Streptococcus pneumoniae.
Figure 18 depicts the periodate and EDC coupling chemistry reactions.
Figure 19 shows how a mono-hapten 8-oligosaccharide tetanus toxoid
conjugate inhibited anti-8 serum binding to a 8 polysaccharide coated ELISA
25 plate.
Figure 20 depicts the IgG antibody isotypes elicited by S. pneumoniae
serotype 8 polysaccharide following i""~.."~ ion with an 8:14 di-hapten-
oligosaccharide-TT conjugate.
- 15- 21~3:733
Figure 21 shows an increased level of IgGl antibody isotype elicited by
polysaccharide following i~ ion with an 8:14 di-hapten-oligosaccharide-
conjugate, typical of a TD response.
Figures 22A and 22B show IgG isotypes elicited from groups of mice
il,,,,,lll~i,~d with 14-polysaccharide and oligosaccharide conjugates with and
without adjuvant.
Summary of the Invention:
In one aspect, the invention provides compositions comprising: a) a size-
separated carbohydrate hapten comprising at least one immlmogenic epitope; and
b) a carrier, wherein said hapten is covalently coupled to said carrier and
wherein said hapten-carrier conjugate is protectively immlmogenic.
1 5
In another aspect, the invention provides methods of making conjugate
compositions comprising: a) cleaving a bacterial polysaccharide into
oligosaccharides so as to preserve immunogenic epitopes on the res--lting
oligosaccharides; b) s~alalhlg the resulting oligosaccharides based on size; c)
selecting those oligosaccharides which contain immlmogenic epitopes based on
inhibition ELISA; d) activating the oligosaccharides selected in step c); and e)coupling the activated oligosaccharides to a purified carrier, wheleill the
res lting composition contains immunogenic epitopes and is protectively
immlln~lgenic .
In a further aspect, the invention provides methods of providing
protective immllni7~tion against a bacterial pathogen comprising ~llmini~tering to
a ll~lllll,~l in need of such treatment an effective amount of the vaccine
composition described above.
21 S3 733
- 16-
In still a further aspect, the invention provides compositions useful for
stim~ ting an immlln~ response to an antigen, said immlmostimlll~tQry
composition comprising an oligosaccharide of S. pneumoniae serotype 8 and a
suitable ph~rm~ceutic~l carrier, wherein said oligosaccharide provides an
5 immllnostimlll~tQry effect.
In a yet further aspect, the invention provides methods of providing
protective immllni7~tion against a bacterial pathogen comprising a-lmini~tering to
a m~mm~l in need of such treatment an effective amount of the composition of
10 the serotype 8 composition described above.
A still further yet aspect of the invention provides methods of aug~ g
an immlmQgenic response to an antigen colllplisillg atlmini~tering an
oligosaccharide of S. pneumoniae serotype 8 which contains an immllnogenic
15 epitope as del~ lilled by inhibition ELISA along with said antigen.
Detailed Des~ tion of the Invention:
This invention relates to improved methods for preparing oligosaccharide-
protein carrier conjugates. The conjugate product may be composed of various
20 haptens or carriers. Mono, di, and multi-hapten conjugates may be plepaled.
Methods to detellllille the presence of immllnogenic epitopes on the hapten or
carrier of the resultant conjugate are described. Such conjugates have utility as
vaccines, therapeutic and prophylactic agents, imml-nomodulators diagnostic
agents, development and research tools.
This invention is particularly suited for developing conjugates as vaccines
to such bacterial pathogens including, but not limited to Streptococcus
pneumoniae, Neisseria meningitidis, Haemophilus influenzae B, Group B
Streptococcus, Group A Streptococcus, Bordetella pertussis, Escherichia coli,
30 Streptococcus mutans, Staphylococcus aureus, Salmonella typhi, Cryptococcus
21 53 733
neoformans, Pseudomonas aeruginosa and Klebsiella pneumoniae. Conjugates
of this invention convert weakly or non-immlmogenic molecules to molecules
which elicit specific immnnoprotective antibody or cellular responses.
Poor immllnP responses to polysaccharide vaccines (thymus independent
antigens, TI) have been observed with high risk groups, such as the elderly and
children under 2 years of age. Several investigators are ~ ing to elicit
thymus dependent (TD) responses to a variety of bacterial polysaccharides using
protein carriers. Integrity of critical immllnogenic epitopes and inconsistency of
10 covalent linkage between the carbohydrate and protein are major limitations with
these conjugate vaccines. The present invention is drawn to the discovery of
coupling technology which gives good reproducibility with respect to the
carbohydrate to carrier ratio of conjugates. This invention also provides
methods to verify the presence of immlmogenic epitopes on and oligosaccharide
15 haptens and hapten-carrier conjugates.
Polysaccharide conjugates elicit non-boostable IgM antibody responses,
typical of TI antigens. The antiserum produced in response to these
polysaccharide conjugates does not have opsonic activity. In the present
20 invention, oligosaccharides prepared by cleavage of polysaccharides from
various bacterial strains are size separated and used to produce mono-hapten
conjugates. These conjugates elicit IgG antibody isotypes with
immllnoprotective, opsonization ability. This antibody response is elicited
without the use of any adjuvant. Thus, the methods of the inventions are ideally25 suited for producing immllnogenic oligosaccharide hapten-carrier conjugates
which utilize weakly or non-immunogenic polysaccharides of various strains.
The presence of immllnogenic epitopes on these oligosaccharides was found to be
critical for eliciting an immnnoprotective response.
. 21S3733
-
- 18 -
The number of bacterial antigens needed to develop efficacious anti-
pathogen vaccines is expanding. However, repeated ~-lmini~tration of tetanus or
diphtheria toxoid (often used as carrier proteins in vaccine compositions and as a
prophylactic measure following trauma) may cause a phenomenon called carrier-
5 inflllce~ epitope suppression. Epitope suppression has been described in thelileldLule with synthetic peptide and saccharide-toxoid conjugates (Gaur et al.,
1990; Peeters et al., 1991). Tmmlln~ responses to a hapten coupled to a carrier
protein can be reduced or absent when the recipient has been previously
illllllllni~ecl with the carrier.
The goal of many researchers is to develop vaccines which elicit
protection to the predo~ all~ bacterial serotypes which cause acute lower
respiratory infection, otitis media and bacteremia in infants, without inducing
carrier suppression. The methods of the invention can be utilized to produce
15 multi-hapten conjugates with optimal immllnogenic epitopes to each bacterial
serotype. These conjugates, which contain lower carrier protein amounts than
traditional conjugates, reduce the occurrence of the carrier suppression
phenomenon. The reduced antigen load possible using these conjugates
",i~ es the antigenic competition observed with traditional conjugates.
Previously, we reported that crystalline bacterial cell surface layers (S-
layers) were useful as carriers for the development of prototype conjugate
vaccines (Malcolm et al., 1993a) and as a means to avoid the carrier
suppression phenomenon (Malcolm et al., 1993b). In our laboratory, we
25 identified several S-layer glycoproteins which elicit non-cross reactive antibody
and cellular responses. Vaccines to a variety of diseases can be developed usingS-layers isolated from various bacterial strains, thereby avoiding carrier
suppression observed with tetanus and diphtheria toxoids. However, S-layers are
difficult to isolate and purify, as well as costly to produce, making them
30 impractical for wide usage as vaccine carriers. The present invention describes
21 ~3 733
- 19-
methods to prepare mono, di and multi-hapten oligosaccharide conjugates which
reduce the amount of carrier n~cess~ry to elicit specific responses, thereby
decreasing the risk of carrier in~ ced epitope suppression, even when tetanus ordiphtheria toxoid is used as the carrier.
One specific application of the technology of the invention is for the
development of effective vaccines for the prevention of pediatric pneumoniae
infections. Another application of the invention is to develop vaccines for
protection to strains of Group B Streptococcus, Group A Streptococcus,
10 Haemophilus influenzae B, Streptococcus pneumoniae and N. meningitidis
prevalent in infant disease, in the elderly or the immllnosuppressed. Other
applications include development of conjugates for eliciting protection to various
bacterial or virus pathogens.
We have found that the use of conditions which cleave specific linkages
(i.e., l - 4 linkages) but leave sugar monosaccharides and other immllnologically
important compounds such as phosphate intact results in improved
immllnogenicity of the resulting conjugates.
We have found that oligosaccharide size and collro~,llalion is important to
m~ximi~e immlm~genicity of conjugate plepdlalion. Dirrelc"l oligosaccharide
sizes are separated from hydrolyzed polysaccharide mixtures and isolated by sizefraction. The monosaccharide content and the relative size of separated
oligosaccharides is measured by, for example, HPLC analysis. Dirrere"l size
25 repeat units are tested using inhibition ELISA. We have found that ELISA
inhibition is directly proportional to the immllnogenicity of the oligosaccharide
preparation and the resultant conjugate.
In particular, oligosaccharides prepared from cleavage of polysaccharides
30 of S. pneumococcus strains 3, 6B, 8, 14, l9F and 23; pneumococcal C-
21 $3733
- 20 -
substance; and N. meningitidis C-polysaccharide have been used in our
laboratory. Preferred repeat units (R.U.) for oligosaccharides are as follows for
some S. pneumococcus serotypes and pneumococcal C-substance:
Serotype 3: 4-8 R.U.
6B: 4-10 R.U.
8: 2-8 R.U.
14: 4-6 R.U.
l9F: 4-10 R.U.
C-substance: 6-10 R.U.
10 Preferred repeat units for N. meningitidis C-polysaccharide is 6-10 R.U.
Creating charged groups on saccharide haptens has been discovered to
facilitate the coupling of the haptens to the carrier. Use of cation or anion
exchange columns is effective in allowing coupling of oligosaccharide to carrier1 5 at a higher sugar to carrier ratio. This provides more hapten per carrier, and
reduces the carrier suppression phenomenon. R~duced fractions of carbohydrate
are used for coupling to carrier.
Another important aspect to produce effective conjugate vaccines is the
20 use of purified carrier. In~u~iLies found in a carrier prepalation may hlle,r~le
with coupling procedures. Aggregates of carrier proteins found in a carrier
p,epa,alion can affect opli~lulll hapten to carrier ratios n~cess~ry to elicit the
desired response. Carriers are generally purified using size exclusion column
chromatography, although any standard method which removes in~ulilies and
25 aggregate may be used.
The coupling reaction time and the amount of oligosaccharide, coupling
reagent and carrier are critical for obtaining an ideal carbohydrate to carrier
conjugate ratio. We have developed methods which quantify carbohydrate to
30 carrier ratios by reproducible assays. Maillle~ ce of pH and temperature
215373~
conditions determined to be optimal during the coupling reaction is also
important to produce an effective conjugate. Likewise, the use of effective
blocking reagents which stop the coupling reaction but do not mask the
immllnogenic groups is important to create effective conjugate compositions.
Use of coupling chemistry which m~int~in~ immllnogenic epitopes on
oligosaccharides/polysaccharides is essential. We have found that EDC and
periodate coupling, as described below may be used for coupling
oligosaccharides to carriers. In addition to direct coupling of sugar to carrier,
10 various linkers may be used to space the saccharide from the surface of the
protein. Appropliate linkers may also provide charged or uncharged moieties as
desired. The immllnogenicity of coupled sugar-carrier compositions is
determined by inhibition ELISA.
Using the methods of the present invention, we have discovered means to
produce di-hapten and multi-hapten conjugates which still m~int~in their
immunogenic epitopes. Conjugates with various oligosaccharide sequences
and/or sizes can be produced. Similarly, conjugates comprising oligosaccharide
and polysaccharide combinations may be synthesized. Such conjugates are able
20 to reduce or elimin~te antigenic competition.
Thus, appropliate conjugate design provides the ability to reduce carrier
in~ ce~1 epitope suppression. Keys in this regard are the identification and use of
immllnogenic oligosaccharide epitopes and more effective coupling of sugar to
25 protein. Binding a larger number of immllnogenic epitopes per protein molecule
means that less carrier is needed to provide protective i~""~ ion.
We have developed methods to quantify immllnnprotective antibody
response to conjugate compositions by isotyping ELISA, bactericidal and
30 opsonization assays. This allows d~telmillation of which conjugates will elicit
21~3733
the appropliate immlmoglobulin isotype response, i.e., IgG isotypes, when used
to protectively immllni7e m~mm~
D~fin;tiQn.c:
The following terms have the following m~ningc when refelellced
herein:
Oligosaccharide means a carbohydrate compound made up of a small
number of monosaccharide units. In particular, oligosaccharides may be formed
by cleaving polysaccharides.
Polysaccharide means a carbohydrate compound cont~ining a large
number of saccharide groups. Polysaccharides found on the outer surface of
bacteria or viruses are particularly useful in the present invention.
Carrier means a substance which elicits a thymus dependent immlln~
response which can be coupled to a hapten or antigen to form a conjugate. In
particular, various protein, glycoproteill, carbohydrate or sub-unit carriers can be
used, including but not limited to, tetanus toxoid/toxin, diphtheria toxoid/toxin,
bacteria outer membrane proteins, crystalline bacterial cell surface layers, serum
albumin, gamma globulin or keyhole limpet hemocyanin.
Immunogenic means causing an immlln~ response. An immllnogenic
epitope means that portion of a molecule which is recognized by the immlm~
system to cause an immllnogenic response.
Hapten means an antigen, including an incomplete or partial antigen
which may not be capable, alone, of causing the production of antibodies. Di-
and multi-hapten, for purposes of this application, refer to compositions
including two (di) or more (multi) oligosaccharide haptens conjugated to carrier.
21~3733
Protectively immllnogenic or immllnoprotective means stim~ tin~ an
immllnP response which prevents infection by pathogen.
Immunostimnl~tory means stimlll~ting or enhancing an immlln~ response
5 to weakly immllnogenic haptens or antigens.
Neonate means a newborn animal, including an infant.
Methodology:
Prep~ration and Separation of Cleaved Polysaccharides:
Polysaccharides, available through American Type Culture Collection,
Rockville, Maryland or by isolation procedures known in the art, were cleaved
into oligosaccharide units using d~ropliate concentrations of chemicals. These
15 chemicals include, but are not limited to trifluoroacetic acid, acetic acid,
hydrofluoric acid, hydrochloric acid, sodium hydroxide and sodium acetate.
Dirre~ time periods and tenlpeldlules may be used depending on the particular
chemistry and concentration and on the resulting oligosaccharide desired.
Commercially available enzymes (e.g., cellulase and ,~-galactosidase) or isolated
20 bacteriophage-associated endoglycans known in the art can also be used to
prepare oligosaccharides from polysaccharides.
Figure 1 shows the repeat unit structures of the polysaccharides used in
the Examples of the invention. Other bacterial and viral polysaccharide are
25 known to those of skill in the art, and may be used in the methods and
compositions of the present invention. Various polysaccharides can be cleaved
including, but not limited to, pneumococcal group antigen (C-substance) and
capsular polysaccharides of serotypes of Streptococcus pneumoniae, Neisseria
meningitidis, Haemophilus inJ?uenzae, Group A Streptococcus and Group B
30 Streptococcus.
21~3733
- 24 -
After cleavage, the resulting oligosaccharide nli~lules are separated by
size using P-10 (fractionation range 1,500 - 20,000 molecular weight), P-30
(2,500 - 40,000 molecular weight) and P-60 (3,000 - 60,000 molecular weight)
BioGel columns. The presence of carbohydrates in the various column fractions
5 is d~lelmilled using phenol-sulphuric or sialic acid assays and thin layer
chromatography (TLC). Carbohydrate-cont~ining column fractions are then
analyzed by HPLC.
The presence of immunngenic epitopes on size-separated fractions of
10 cleaved polysaccharides is d~le.lllil1ed by inhibition ELISA, as described below.
If a preparation does not result in oligosaccharide fractions which inhibit in the
ELISA test, cleavage procedures may be modified by ch~nging enzymes or
chemicals, molarity, reaction time or l~ el~lule in order to produce
immlmogenic epitopes.
1 5
D~le,lllhlation of Tmmuno~enic Epitopes in Oligosaccharide Preparations:
The presence of immlmogenic epitopes in column fractions is confirm~cl
by inhibition ELISA and phosphorous assay as set forth in the Examples section.
Oligosaccharide fractions cont~ining immllnogenic epitopes (defined as those
20 which produce at least about a 50% reduction in O.D.40s at 12.5 llg
concentration) are selected for coupling to carrier.
Couplir~ to C~rrier:
The oligosaccharide or polysaccharide to be used for coupling to carrier
25 is acidified or reduced in p-epalalion for EDC or periodate oxidation coupling.
For example, the oligosaccharide plepal~lion may be reduced using a RexynTM
101 (H) organic acid cation exchange column to acidify the sugar for EDC
coupling. Similarly, sugars may be reduced using standard methods for
periodate oxidation coupling. When pl~alillg di-hapten or multi-hapten
21 ~3 73~
-
- 25 -
conjugates, each oligosaccharide is activated individually for EDC or periodate
conjugation.
Preferred di-hapten oligosaccharide conjugates include: 3:8-TT, 6:8-TT,
5 6:14-TT, 8:14-TT, 8:19-TT, 8:23-TT and 14:19-TT.
Carrier:
Various protein, glycoprotein, carbohydrate or sub-unit carriers can be
used, including but not limited to, tetanus toxoid/toxin, diphtheria toxoid/toxin,
10 bacteria outer membrane proteins, crystalline bacterial cell surface layers, serum
albumin, gamma globulin or keyhole limpet hemocyanin. In the specific
examples of this invention, tetanus toxoid was used as the carrier. Tetanus
toxoid l)r~al~lions routinely contain aggregates and low molecular weight
hll~ulilies. Purity of carrier is essential for obtaining consistency with coupling
15 reactions, size exclusion chromatography is used to obtain a purified carrier preparation.
Size separated, immllnogenic epitope-cont~ining oligosaccharides are
coupled to purified carriers by carbodiimide (EDC) or periodate activation, using
20 the procedures described in the Examples section. Any free hapten
oligosaccharides are separated from hapten-carrier conjugates by column
chromatography. The carbohydrate to protein ratio of conjugates is delellllilledby phenol sulfuric or sialic acid and Lowry protein assays. Typically, conjugates
prepared by EDC coupling have a carbohydrate to carrier ratio of 1:2, while
25 conjugates prepared using periodate oxidation coupling have carbohydrate to
carrier ratios ranging from 1:5 to 1:10.
Delellllillalion of Tmmllno~enic Epitopes on Conju~ates:
As stated previously, integrity of critical immunogenic epitopes is a
30 problem with previously known conjugation technologies. In the present
21~373~
- 26 -
invention, the ELISA inhibition assay is used to determin~ the potential
immunogenicity of various conjugates produced by our conjugation procedures.
We have found that conjugates which demonstrate inhibition in this assay (at
least about 50% reduction in O.D.4~,5 at 6.25 ~lg concentration) using the
5 methods set forth in the Examples, provide protective immlmc)genicity when used
as a vaccine in m~mm~ . Thus, this assay is used to screen for useful conjugate
compositions.
Tmml1ni~tion to Flicit Tmmunoprotective Antibody Respon.~es:
Typically, mice are immuni7ed on day 0 (1-primary immuni7~tion) day 7
(2-secondary immuni7~tion) and day 28 (3-tertiary i"",~."~ ion) by
subcutaneous injection (100~11 into 2 flank sites) with antigens (polysaccharide-
conjugates, oligosaccharide-conjugates, uncoupled polysaccharide or
oligosaccharide, or uncoupled tetanus toxoid) at doses of 0.1, 0.5, 1, 2.5 and 515 ,ug, based on carbohydrate content for EDC conjugates and protein content for periodate conjugates.
Antigens were diluted to various doses in 0.9% NaCl and mice injected
with 0.9% NaCl were used as negative controls. Mice were bled 7-10 days post
20 -2 and 3 immuni7~tion to collect serum to assay immunoprotective antibody
responses. A typical i~ ni~;.lion schedule is shown in Table 1 for S.
Pneumoniae serotype 3 polysaccharide and oligosaccharide-tetanus toxoid
conjugates prepared using EDC coupling.
Various other i~ lni7i.tion schedules are effective, including: day 0 (1),
day 14 (2) and day 44 (3); and day 0 (1), day 30 (2) and day 60 (3).
The conjugates of this invention may be used as classical vaccines, as
immunogens which elicit specific antibody production or stim~ te specific cell
30 mediated immlmity responses. They may also be utilized as therapeutic
2153733
- 27 -
modalities, for example, to stim~ te the immlln~ system to recognize tumor-
associated antigens; as immlmomodulators, for example, to stimlll~te
lymphokine/cytokine production by activating specific cell receptors; as
prophylactic agents, for example, to block receptors on cell membrane
5 preventing cell adhesion; as diagnostic agents, for example, to identify specific
cells; and as development and/or research tools, for example, to stimlll~te cells
for monoclonal antibody production.
Detellllhla~ion of Response:
As previously discussed, antibody responses to TI and TD antigens differ.
In the mouse, the response to a polysaccharide (TI) antigen is usually composed
of a one-to-one ratio of IgM and IgG. In general, IgG isotypes are restricted,
with IgG3 being over-expressed in anti-polysaccharide serum. IgA isotypes may
also be present. TI antigens elicit antibodies with low affinity and immllnologic
15 memory is not produced.
With TD antigens, increased secondary IgG antibody responses (an
~n~mn~stic response) are found, with a higher IgG to IgM ratio. Marked levels
of IgA are usually not present. The TD antigen elicits a heterogeneous IgG
20 isotype response, the predominant isotype being IgGl. IgGza and 2b isotypes can
be expressed, while the IgG3 isotype level is usually relatively low. TD antigens
elicit immllnologic memory and antibody affinity increases with i~ i7~ions.
Thus, analysis of the immlmnglobulin isotypes produced in response to conjugate
~tlrnini~tration enables one to determine whether or not a conjugate will be
25 protectively immllnogenic.
We have found that the conjugates of the present invention induce a
response typical of TD, rather than TI antigens, as measured by direct and
isotyping ELISA and opsonization assay.
21S3733
- 28 -
Conjugates prepared using our EDC coupling methods elicited better
antibody responses than conjugates prepared by periodate activation. Doses of l
~lg were most immunogenic. Oligosaccharide-conjugates plcpalcd with
diphtheria toxoid carriers elicited antibody responses similar to the responses
5 elicited with the oligosaccharide-tetanus toxoid conjugate.
As described previously, several investigators have ~llellll)t~d to increase
immunogenicity and elicit thymus-dependent antibody protection by coupling
polysaccharide material to tetanus and diphtheria toxoids. Results indicate that10 these conjugates are only slightly more imm-lnogenic than uncoupled capsular
polysaccharide (CPS). One possible explanation for this may be that pertussis,
diphtheria and tetanus toxoids (in allllllilllllll salt adjuvant) are often ~lmini~tered
as a prophylactic four dose i~ lni~tion regime to infants. This regime may
tolerize the infant, making the infant incapable of mounting a protective antibody
15 response to a hapten/antigen coupled to these toxoid carriers (carrier
suppression). Another possible reason for failure to induce protection may be
structural. Protein carriers elicit and augment the immlmP response to haptens,
but in the case of CPS-protein conjugates, the CPS portion is a relatively largeTI antigen. The immlm~ system may not recognize the CPS-protein as a
20 conjugate, but simply as two distinct entities, resulting in a thymus-independent
response to the CPS and a thymus-dependent response to the carrier.
This appears to be the case in our studies, as shown in Table 2. The
immlmP. system recognizes the polysaccharide of our polysaccharide-tetanus
25 toxoid (TT) conjugate as a TI antigen. The potential TD inducing capability of
the carrier with respect to antibodies to the polysaccharide is not observed. Wepostulate that the immllnngenic epitopes of the carbohydrate haptens
(oligosaccharides) must be in close proximity to the TD inducing epitopes of thecarrier in order to convert a TI response to a TD response.
2153733
- 29 -
We have also used linker arm technology to prepare conjugates. We have
used, for example, 6-amino-n-hexanoic acid as a linker. The resulting
conjugates were found to be less effective in eliciting antibody responses than
conjugates prepared by directly coupling EDC activated oligosaccharide haptens
5 to carriers. This finding supports our hypothesis that close hapten to carrier proximity is needed to elicit TD responses.
We have also developed methods to dele~ e the level of
immllnoprotective antibody elicited by the conjugates of the present invention
10 using bactericidal or opsonization assays. These tests have shown that the
conjugates of the present invention are effective in eliciting protective antibodies,
as measured by these assays.
As discussed previously, the epitope-carrier suppression phenomenon has
15 been observed by other researchers and in our laboratory with the S-layer carrier
studies (Malcolm et al., 1993b). Our multi-hapten conjugates will reduce or
circumvent this suppression, because these conjugates will contain greater mass
of immllm)genic epitope per molecule of carrier than conventional conjugate
vaccines. With our conjugates, the immlmP system will not be "overchallenged"
20 by the carrier. For example, a tri-hapten conjugate prepared by methods of this
invention will require only three injections to elicit specific immllnP responses to
three different target pathogens. In contrast, using conventional monohapten
conjugates, one would need to ~lmini.cter nine injections to elicit similar
responses. This means three times the amount of protein would be required.
Further, i~ tion regimes convert an anti-polysaccharide TI
response to a TD response can be designed using the conjugates of the present
invention. Economical initial exposure to polysaccharide (e.g., using
Pneumovax 23) followed by a single ~(1mini~tration of a conjugate of the present30 invention would induce IgG antibody levels (an ~n~mn~stic response). Such an
2IS3733
- 30 -
i""",."i~lion regime would not induce carrier suppression. In such cases, the
imml-n-~ system initially e~luc~ted to various carbohydrate epitopes and antigens
(a TI response) would be in(l~1ce~1 by multi-hapten conjugates to elicit stronger
immllnngenic responses to pathogens frequently causing disease in specific
5 population groups (e.g., serotypes 1, 3, 4, 6, 9, 14, 18, 19 and 23 in infants).
Pharm~^e~lt~ C~ o~ ns:
To elicit antibodies to specific pathogens and/or various carbohydrate
moieties the conjugates of the invention may be ~flmini~tered by various delivery
10 methods including intraperitoneally, in~ldu"lsc~ rly, intradermally,
subcutaneously, orally or nasally.
The formulation of the compositions of the present invention may include
suitable ph~rm~ceutical carriers. The conjugates of the invention are
15 immllnngenic without adjuvant, however adjuvants may increase
immunnprotective antibody titers or cell mtq/li~tecl i~l~u~u~iLy response. Such
adjuvants could include, but are not limited to, Freunds complete adjuvant,
Freunds incomplete adjuvant, alumilliulll hydroxide, dimethyldioctadecyl-
ammonium bromide, Adjuvax (Alpha-Beta Technology), Inject Alum (Pierce),
20 Monophosphoryl Lipid A (Ribi Tmmnnnchem Research), MPL+TDM (Ribi
Immunochem Research), Titermax (CytRx), toxins, toxoids, glycoproteins,
lipids, glycolipids, bacterial cell walls, subunits (bacterial or viral), carbohydrate
moieties (mono-, di-, tri- tetra-, oligo- and polysaccharide), various liposome
formulations or saponins. Combinations of various adjuvants may be used with
25 the conjugate to prepare the immnnogen formulation.
Exact formulation of the compositions will depend on the particular
conjugate, the species to be i~ e~l and the route of ~lmini~tration.
21~3733
Such compositions are useful for i~ any animal susceptible to
bacterial or viral infection, such as bovine, ovine, caprine, equine, leporine,
porcine, canine, feline and avian species. Both domestic and wild anim~lc may
be immnni7~1. Humans may also be il~ kd with these conjugate
5 compositions.
The route of a(lmini.ctration may be any convenient route, and may vary
depending on the bacteria or virus, the animal to be i~ e~l and other
factors. P~ellLelal ~flminictration, such as subcutaneous, h~ .lcc~ r, or
10 intravenous a(lminictration, is prefelled. Subcutaneous a~lminictration is most
pler.,ll~d. Oral atlminictration may also be used, including oral dosage forms
which are enteric coated.
The schedule of a~lminictration may vary depending on the bacteria or
15 virus pathogen and the animal to be i~"",l~i7e~1. Animals may receive a single
dose, or may receive a booster dose or doses. Annual boosters may be used for
continued protection. In particular, three doses at days 0, 7 and 28 are pleÇ~.red
to initially elicit antibody response.
The following examples are not intended to limit the scope of the
invention in any manner.
Examples
F7~ml)le l:
Pl~paldLion and Separation of Polysaccharide Hydrolysates
Figure 2 shows the separation profile of Streptococcus pneumoniae
serotype 8 capsular polysaccharides through a BioGel P-l0 column after acid
hydrolysis (0.5 M trifluoroacetic acid, 100C, 20 Illill~ s) resulting in
discernible oligosaccharides of one to eight repeat units. Numbers one to eight
- 32 - 21 ~3 733
correspond to the number of repeat units found in each peak, peak nine contains
oligosaccharides of greater than eight repeat units. Oligosaccharides derived
from hyaluronic acid were used to standardize the chromatographic system.
The relative size of the repeat units in peaks 1, 2, 3 and 4 were measured
by HPLC analysis (Figure 3). The HPLC retention times of glucose, M-3
maltotriose, M-7 maltoheptose, and M-10 malto-oligosaccharide (Sigma
Chemical Co.) used as standards to delelnlille relative size of various
oligosaccharide repeat units is shown in Figure 4. Monosaccharide content of
10 the repeat structure was established by further hydrolysis of the oligosaccharide
repeats with 2.0 M trifluoroacetic acid (TFA) at 100C for 2 hours. An example
of the retention times of ribitol, rhamnose, galactose, fucose and mannose
monosaccharide standards used to determine carbohydrate content of the
hydrolysed repeat unit is shown in Figure 5. The chemical structure of one
15 serotype 8 repeat unit was determine to be ,B-glucose (1 ~ 4) ,~-glucose (1 ~ 4)
a-galactose (1 ~ 4) aglucuronic acid (1 ~ 4) by GC-MS and NMR analysis.
This corresponds to the repeating unit structure cited in the lileralule (Jones and
Perry 1957).
Figures 6 - 10 are examples of separation profiles of S. pneumoniae
serotypes 6B, 14, l9F and 23F polysaccharide hydrolysates (TFA, acetic acid or
hydrofluoric acid) passed over P-10, P-30 or P-60 BioGel columns.
Figure 11 shows the separation of an enzyme cleaved polysaccharide
(serotype 8 cleaved by cellulase). The separation of C-substance
oligosaccharides is shown in Figure 12.
2153733
Example 2:
Inhibition FT T~SA to Determine Immunogenic F,r~itopes of Oli~osacch~ride
Preparations
The basic procedure utilized for inhibition ELISA to test for the presence
5 of immllnogenic epitopes on oligosaccharide ple~al~lions and oligosaccharide or
polysaccharide-conjugates was as follows:
1. Coat 96 well EIA plates (NUNC) with 1 ~lg well of the antigen (Ag)
using 0.05 M NaCO3 coating butter (100 ~ll/well), incubate at 4C overnight.
2. On the same day, prepare inhibiting Ag tubes (e.g., various
1 0 oligosaccharide hydrolysates) using 1 x PBS - 0.01 % Tween 20 as diluent.
- Make a 7 fold serial dilution in the tubes (starting from 25 ~lg/well to
0.391 ,ug/well in triplicate), the total volume in each tube should be 175 ~11 after
serial dilution.
- Prepare 1: 1000 dilution of anti-serum of a specific type (e.g., Diagnostic
1 5 anti-serum 14 that has been raised in rabbits, Statum Seruminstitut), in 1 x PBS
+ Tween.
- Add 175 ~11 of this solution to each tube. Total volume in each tube is
now 350 ~1. Incubate the tubes at 4C overnight.
3. Next day, block the EIA plates with 100 ~ll/well of blocking buffer (1 x
20 PBS + 1 % BSA), incubate at room temperature for 1 hour.
4. Flick off the plates and Llall~re~ content of each tube to the wells (100
~ll/well, incubate at room temperature for 2 hours.
5. Wash the plates 3 times with wash solution (0.01 % Tween+ 1 x PBS).
6. Prepare 1: 1500 dilution of Goat-anti-rabbit (or anti-species of serotype
25 specific serum used in Step 2) IgG Alkaline Phosphatase conjugate (TAGO) in 1x PBS + 1 % Tween buffer (100 ~ll/well). Incubate at room temperature for 2
hours.
7. Wash the plates 4 times with wash solution, flick off excess liquid.
8. Dissolve Alkaline Phosphatase substrate tablets (# 104 - Sigma) in the
30 DEA (diethylen~min~-) buffer pH=9.98, 5 ml/tablet, 100 ~l/well.
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9. Incubate the plates in the dark and read the Absorbance at 405 nm
wavelength every 15 minutes.
Various commercial and laboratory prepared antiserum can be used in this
5 assay, including, but not limited to, serum produced in mice, rat, rabbit, goat,
pig, monkey, baboon and human.
Figure 13 shows the inhibition ELISA results using a mouse antiserum to
Streptococcus pneumoniae serotype 8 oligosaccharide protein carrier conjugate
10 (2-4 repeat units coupled using EDC to TT). Inhibition was tested with type 8oligosaccharides (0.5 M TFA, 100C, 20 minute preparation) of 1, 2, 3, 4, 6, &
8 + repeat units, and with type 8 polysaccharides. From these results, it can beseen that the 1 repeat unit (a 4 monosaccharide chain) does not contain an
immlmogenic epitope. The 2 repeat unit (8 monosaccharide chain) was capable
15 of inhibiting antibody binding to the ELISA plate, indicating that it contains an
immllnogenic epitope. The molecular weight of repeat unit 2 was d~Le~ ed to
be 1365 by FAB-MS analysis. This correlates well with the theoretical
molecular weight of 8 saccharides. Repeat units of 3, 4, 6, 8 + and the whole
polysaccharide also inhibited antibody binding to the ELISA plate, again
20 indicating that immllnogenic epitopes were present in these
oligo/polysaccharides .
Table 3 demonstrates similar results found using a rabbit anti-S.
pneumoniae serotype 8 specific serum (Statems S~lulllin~LiLuL). Repeat unit 1 did
25 not markedly inhibit binding; repeat units 2, 3, 4, 5, 6, 7, 8+ and whole
polysaccharide inhibited binding.
Inhibition ELISA was also used to de~ellllille the presence of
immunogenic epitopes on oligosaccharides prepared using dirrelellL hydrolysis
30 procedures on various polysaccharides. Table 4 shows results with methods used
`_ 21 53733
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by the prior art, for example, Porro C~n~ n Patent No. 2 052 323 to hydrolyse
S. pneumoniae serotype 6 polysaccharide (0.01 M acetic acid, 100C, 30 hours).
Whole polysaccharide blocked binding at low antigen concentration (effective at
0.39 ~g concentration) while the acetic acid hydrolysate did not. Note that we
5 could not size separate the hydrolyzed preparation because it was "caramelized."
We discovered that dirrelell~ hydrolysing agents (e.g., TFA) and reduced
time and tempeMture produced oligosaccharides with more immllnogenic
epitopes, as shown in Table 5. A 0.5 M TFA, 70 C 1 or 2 hour hydrolysate
1 0 effectively inhibited antibody binding at a 3.13 ~lg concentration, a 4 hourpreparation did not. Tables 6 and 7 also illustrate the effect of time for
plepalillg 6B oligosaccharides with or without immllnogenic epitopes. A 2 hour
acetic acid preparation blocked antibody binding (at 3.13 llg concentration), the
24 and 48 hour prepalations did not. Similarly, a 1.5 hour TFA preparation
1 5 more effectively blocked binding than a 3 hour preparation.
As shown in Table 8, 0.5 M TFA hydrolysis of S. pneumoniae serotype
14 at 70C for 7 hours, as disclosed in the prior art (Porro, C~n~ n Patent 2
052 323), is not ~refelled. l~ed~1cecl molar concentrations of TFA (e.g., 0.1 M)20 is better for prepalillg immunogenic 14 oligosaccharides.
Table 9 illustrates the importance of selecting oligosaccharides which
contain immllnogenic epitopes for coupling to carrier. The 3 repeat unit
structure of serotype 14 oligosaccharide could not inhibit antibody binding, the 4
25 and 8 repeats, however, contain the immlln()genic epitopes and effectively
blocked antibody binding.
Table 10 shows the effect of hydrolysate concentration and reaction time
for pl~alillg 14 oligosaccharides cont~ining immllnogenic epitopes.
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Tmmllnogenic epitopes were conserved by a TFA 7 hour hydrolysis, but
destroyed when hydrolysed for 24 hours.
Table 11 illustrates the importance of using optimal heat conditions for
5 producing 19F oligosaccharides cont~ining immlmogenic epitopes. Tmmlmogenic
epitopes were destroyed by HCl hydrolysis at room temperature, but m~int~in~d
when hydrolysis was performed at 70C.
As shown in Table 12, poor inhibition of antibody binding was observed
1 0 with 0.25 M TFA, 70C, 3 hr hydrolysates of 23F polysaccharides, (Porro,
C~n~ n Patent 2 052 323). Table 13 demonstrates the effect of time on the
generation of immllnogenic 23- oligosaccharides. Oligosaccharides produced by
0.1 M TFA hydrolysis, 70C for 3 hours inhibited antibody binding,
oligosaccharides prepared by hydrolysis for 5 hours did not inhibit. Table 14
1 5 demonstrates the presence of immlmogenic oligosaccharides after 0.5 M TFA
hydrolysis at 70C for 15 minutes or with 5 M acetic acid at 70C for 5 hours.
These hydrolysates effectively inhibited to 0.78 ~g concentration.
Table 15 demonstrates the utility of the inhibition ELISA to recognize
20 immunogenic oligosaccharides of Neisseria meningitidis serotype C.
Hydrolysates prepared with NaOAc, blocked antibody binding as effectively as
the whole polysaccharide.
Example 3:
25 Acidification of Carbohydrate Moieties for Carbodiimide Couplin~
A Rexyn 101 (H) organic acid cation exchange column (Fisher Scientific)
was prepared and washed with dH20. Polysaccharide or oligosaccharide samples
dissolved in dH20 (pH neutral) were run over this column and collected at a rate30 of one drop per six seconds. Acidification was confirm~d by pH colour-fixed
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intli~ator sticks. Excess dH20 was used to wash the column. Acidified fractions
were pooled and lyophilized for use in coupling reactions.
Figure 14 depicts a TFA cleavage between ,~-D-Glcp (1 ~ 4),B-D-Gal of
5 an oligosaccharide structure resulting in the formation of an aldehyde and
hydroxyl group. Further oxidation of the aldehyde results in a carboxyl group.
When this material is passed through a cation exchange column, a COO~ group
results.
10 Fx~mple 4:
Couplin~g Procedures and Quantification Assays
Carbodiimide (EDC) Couplin~ Procedure
A 1:1 weight ratio of ion charged carbohydrate (polysaccharide or
15 oligosaccharide) sample (e.g., 3 mg) and EDC (3 mg) was dissolved in 2 mls of 0.1 M KH2PO4, a pH of 4.5 was m~int~in~d with lN NaOH or HCl. This
mixture was stirred for 1 hour at room temperature. Carrier (3 mg) was added
to the EDC activated carbohydrates and then stirred for 4 hours at room
temperature. This reaction was stopped by the addition of 200 ~11 of 10%
20 ammonium bicarbonate, the mixture was then further stirred for 1 hour at room temperature.
The resultant conjugate was dialysed against dH20 overnight using 50,000
molecular weight cut off (MWCO) dialysis tubing.
Conjugates were lyophilized and then assayed by Lowry protein, phenol-
sulfuric acid, sialic acid and phosphorous assays for composition (methods
described below). Typically, conjugates prepared with this coupling methods
have a carbohydrate to carrier ratio of 1:2.
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Phenol-Sulfuric Acid Assay for Quantification of Carbohydrates
Reagent: 5% phenol solution (5.5 mL liquid phenol (90%) added to 94.5 mL
distilled water).
5 Standard: Glucose 1 mg/ml stock solution. Prepare 2 to 60 ,ug/200 ,ul sample
buffer for standard curve.
Procedure: (Adapted from: Handbook of Micromethods for the Biological
Sciences. Keleti, G. & W.H. Lederer (eds). 1974. Van Nostrand Reinhold Co.,
New York.)
1 0 1. Place 200 ~11 samples into very clean tubes.
2. Add 200 111 phenol reagent.
3. Rapidly add 1 mL concentrated sulfuric acid.
4. Vortex well.
5. Let stand at room temperature for 30 minutes.
1 5 6. Color is stable at room temperature for 2 to 30 hours.
7. Read absorbance at 490 nm: blank with tube cont~ining H20 only as
sample in # 1.
Q~1~ntit~tive Estim~tion of Si~lic Acid
20 Reagents:
a. 6 gram of A12(SO4)3 . 18 H20 dissolved up to 20 ml with distilled water.
b. 1 gram para-dimethylaminobenzaldehyde dissolved up to 20 ml with 6 N
HCl. (Store in a dark bottle in the refrigerator).
Standard: N-acetyln~ul~llh~ic acid at 0, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45
25 and 50 ~lg/~ll total volume is 350 ~11. Use distilled water to make up to 350 Method:
1. 200 ~11 of sample in duplicates. Make up to 350 ~1 with distilled water.
2. Add 700 ,ul of reagent A to each tube. Shake.
3. Add 350 ~11 of Ehrlich reagent B.
30 4. Cover all tubes with marbles.
. ~ 2153733
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5. Heat the tubes at 100 C for 30 minutes using Pierce Heating modules.
6. Cool the tubes rapidly to room temperature in an ice bath.
7. Read optical density at 530 nm wavelength.
5 Phosphorous Assay
Reagents: a. 2.5 % ammonium molybdate; b. 10% ascorbic acid; c. 70%
perchloric acid; and d. 1 mM sodium phosphate standard.
Procedure: (Adapted from: Rouser, G., Siakotos, A. N. and Fleischer, S. 1966.
Lipids 1:85-86)
1 0 1. Place samples and standards (0, 25, 50, 100 and 200 ml; 25 to 200
nmoles) into clean tubes.
2. Dry samples in a heater block at 180C for 5 minutes in the fume hood.
3. Add 450 ml perchloric acid to each tube, cover each tube with a marble
and heat at 180c for 30 - 60 minutes.
1 5 4. Add 2.5 mL d.H20. after tubes have cooled.
5. Add 0.5 ml ammonium molybdate and vortex imm~ tely.
6. Add 0.5 ml ascorbic acid and vortex immediately.
7. Place tubes in 95c water for 15 minutes.
8. Read absorbance at 820 nm after tubes have cooled.
20 9. Samples can be left for several hours before being read.
T owry Protein Assay
Reagents:
a. 2% (w/v) Na2CO3 in 0.1 M NaOH (1 L)
25 b. 0.5% CuS04 in 1% sodium citrate (100 mL)
c. Folin-Ciocalteu phenol reagent (2X)
d. Bovine serum albumin (1 mg/mL)
Procedure:
1. Prepare standard curve which consists of: 0, 12.5, 25, 50, 100 and 200
30 mg of BSA in a final volume of 200 ~lL.
_ 21~373~
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2. Bring unknown protein samples to 200 mL with d.H20.
3. Mix reagents A and B 50: 1 (v/v) and add 2 mL to each sample.
4. Vortex and let stand at room t~lllpel~Lule for 10 minutes.
5. Dilute Folin-Ciocalteu phenol reagent 1: 1 with d.H20 and add 200 mL to5 each sample.
6. Vortex and let stand at room temperature for 30 minutes.
7. Read absorbance at 660 nm.
Periodate Oxidation Couplin~ Procedure
1 0 Samples of polysaccharide or oligosaccharide (e.g., 3 mg) were dissolved
in 3 ml of freshly prepared 60 mM sodium meta-periodate in 50 mM sodium
acetate. This preparation was then stirred overnight at 4C. Ethylene glycol (300
~l) was then added to stop the reaction, this mixture was subsequently stirred at
room temperature for 1 hour and then lyophilized. Samples dissolved in 1.5 ml
1 5 of 0.03 M ammonium bicarbonate (pH = 8.0) were run over a P-2 Bio-Gel
column. The phenol-sulfuric acid or sialic acid assays were used to del~ line
fractions cont~ining the periodate reduced form of the samples, which were
subsequently lyophilized.
Figure 15 shows the separation of a reduced polysaccharide (23 valent
polysaccharide vaccine-Pneumovax~ 23, Merck, Sharp and Dohme) fraction.
Figures 16 and 17 demonstrate separation of reduced oligosaccharides of
serotypes 6B and l9F of Streptococcus pneumoniae, respectively.
Three mg of reduced saccharide and 3 mg of carrier were dissolved in 3
mls of 0.1 M sodium tetraborate decahydrate, pH 8.9. Sodium
cyanoborohydride (H+ source) was then added to this mixture and stirred for 48
hours at 50C. This reaction was stopped by adjusting the pH to 3 - 4 with 80%
acetic acid. This conjugate was then dialysed for 48 hours against dH20 (2 - 3
30 dH20 changes) using 50,000 MWCO dialysis tubing.
- 21S3733
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The conjugate was lyophilized, and the composition of the conjugate
determined by Lowry protein assay, phenol-sulfuric, sialic acid and phosphorous
assays. Typically, conjugate prepared using this coupling method have
carbohydrate to carrier ratios of 1:5 to 1:10.
Figure 18 depicts the periodate and EDC coupling chemistry reactions.
Example 5:
Conjugate Carriers
Example 4 describes methods used to produce immlln~genic
oligosaccharide/polysaccharide conjugates from weakly or non-immunogenic
polysaccharides .
Tetanus toxoid was purified for use as a carrier by column
chromatography. This purified toxoid elicited high levels of IgM (e.g., SO~lg/mlmouse serum) and IgG isotypes (e.g., IgG, 100 llg/ml of serum; IgG2a, 38 ~g/ml
of serum; IgG2b, 68 llg/ml of serum; and IgG3, 105 ~g/ml of serum).
20 Example 6:
Delel",i~-~tion of TmmllnQgenic F.pitopes on Oligosaccharide/Polysacch~ride
Conju~ates:
The inhibition ELISA as described in Example 2 was used. The
presence of immllnogenic epitopes on a mono-hapten 8-oligosaccharide tetanus
25 toxoid conjugate was confirmed by inhibition ELISA. This conjugate inhibited
anti-8 serum binding to a 8 polysaccharide coated ELISA plate (Figure 19). Free
tetanus toxoid did not inhibit binding. The presence of immllnogenic 8
oligosaccharide on di-hapten 6:8; 14:8 and 19:8 conjugates was also shown.
This figure illustrates the reproducibility of our coupling procedures, as the 8-
21~3733
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mono-hapten and di-hapten conjugates equally blocked antibody binding,
indicating that each conjugate contained equivalent amounts of 8 oligosaccharide.
Table 16 shows results of inhibition ELISA when 6B polysaccharide, 6B
5 oligosaccharides, a 6B:8 di-hapten-oligosaccharide tetanus toxoid conjugate ortetanus toxoid alone was used as inhibiting antigens. Tetanus toxoid did not
inhibit binding of anti-6B serum to a 6B-polysaccharide coated ELISA plate.
Free 6B-oligosaccharide or polysaccharide did inhibit binding. The 6B:8 di-
hapten-oligosaccharide-TT conjugate also inhibited binding. This confirms the
10 presence of immllnogenic 6B epitopes on the 6B:8 di-hapten-TT conjugate.
Similarly, a 14:8-di-hapten-TT conjugate inhibited anti-14 serum binding,
demonstrating the presence of serotype 14 immllnogenic epitopes (Table 17).
Note that at high concentrations, there was non-specific inhibition by TT alone.15 We have found that this is an artifact of anti-14 in this assay.
Various oligosaccharide fractions of a 23F hydrolysate were coupled to
TT. All contained immnnngenic epitopes of the 23F serotype as shown in Table
18.
The immnnogenic epitopes of N. meningititli.~ oligosaccharides (NaOAc
preparation) were similarly m~int~in~d when coupled to tetanus toxoid (see Table19).
25 Example 7:
Detellnillillg Antibody Isotype Levels Elicited by Thymus Independent (TV and
Thymus Dependent (TD) Antigens
The basic procedure to measure antibody isotype levels is as follows to
quantify IgM, IgG and IgA isotypes elicited by various conjugates:
21S3733
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1. Coat EIA plates (NUNC, IMMUNOSORB) with 1 mg/well of Ag in 0.05
M sodium carbonate/sodium bicarbonate buffer pH-9.5, 100 ,ul/well.
2. Incubate at 4C overnight.
3. Next day, block plates with 100 ml well of blocking buffer (1 x PBS +
5 1% BSA). Tnrl1bate at room temperature for approximately 1 hour.
4. Prepare 1:25 dilution mouse serum in working-buffer (1 x PBS + 0.1%
Tween). Add 100 Ill/well into the approl)liate well, incubate at room
temperature for 2 hours.
5 . Wash plates 3 x with washing buffer ( 1 x PBS + 0.05 % Tween). Flick
10 off excess liquid by tapping the plates on the bench top.
6. Prepare 1:2 dilution of EIA Grade Mouse Type (Rabbit Anti-Mouse,
IgM, IgG~, IgG2a, IgG2b, IgG3 and IgA, Bio-Rad) in working buffer at 100
~l/well. Tnrllbate at room temperature for 2 hours.
7. Wash plates 3 x with washing buffer.
1 5 8. Prepare 1:1500 dilution of Goat-anti-Rabbit IgA Alkaline Phosphatase
conjugate (TAGO) in working buffer at 100 ml/well. Incubate at room
l~lll~elaLu~ for 2 hours.
9. Wash plates 4 x with washing buffer.
10. Prepare enzyme substrate using Sigma # 104 ALkaline Phosphatase
20 Substrate tablets (one tablet/5 mls of 10% diethanolamine substrate buffer), 100
Ill/well. Tn~llbate at room temperature in the dark and read every 30 minl-tes at
405 nm wavelength.
11. Convert absorbance readings to mg antibody/ml serum using dose-
response curves geneldL~d from ELISA responses, of the rabbit anti-mouse
25 isotype antibodies to various concentrations of mouse class and subclass specific
immunoglobulin (Zymed Labs. Inc.).
Table 2 shows the antibody elicited in mice when immllni7Pcl with S.
Pneumoniae serotype 8 oligosaccharide and polysaccharide conjugates. Only the
30 8 oligosaccharide-conjugate elicited IgG antibodies of all isotypes, the
~1~373~
- 44 -
unconjugated oligosaccharide was not immlln~genic, the polysaccharide and the
polysaccharide-conjugate elicited antibody isotypes typical of TI responses
(mainly IgM, IgA, and IgG3 isotypes). Adjuvant was not n~cess~,ry to elicit the
IgG isotypes with our oligosaccharide-tetanus toxoid conjugate. Conjugates
5 comprising relatively small oligosaccharides, haptens of 2 - 4 repeat units (8 - 16
saccharides), elicited the best antibody responses as measured by direct ELISA.
Direct FT T~SA Protocol
1. Use NUNC Maxisorp Immunoplate.
10 2. Dilute coating antigen to 1.0 mg/100 ml in carbonate-bicarbonate buffer.
Use glass tubes as antigen will stick to plastic.
3. Add 100 ml to each well of plate. Store overnight at 4C.
4. Wash 3 x in PBS-.05 % Tween. Shake out excess PBS by tapping on
Kimwipes/paper towels.
15 5. Add 100 ml/well of blocking agent (1 x PBS - 1% BSA). Incubate for 60
minutes at room temperature.
6. Wash 3 x as in Step 4.
7. Add 100 ml/well of test antibody appropliately diluted in PBS - .01%
Tween. Incubate for 90 minlltes at room temperature.
20 8. Wash 3 x as in Step 4.
9. Dilute ~lk~lin~ phosphatase conjugated anti-mouse Ig (TAG0 Cat # 4653)
in PBS-Tween 1/1500. Add 100 Ill/well and incubate for 90 minutes in the dark.
10. Wash 3 x as in Step 4.
11. Add 100 ml/well Sigma 104 Phosphatase Substrate (disodium-p-
25 nitrophenyl phosphate tables). Add 2 tablets (5 mg/tablet) of substrate to 10 mLdiethanolamine buffer. Keep in dark as substrate is inactivated by light.
12. Incubate in dark at room temperature. The development of the reaction
varies depending on the antibody. Absorbance can be read on the Microelisa
Auto Reader (405 nm) at approximate 30 minlltes intervals.
21 53 733
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Results in Table 20 show a comparison of IgG1 and IgG3 levels in mice
immuni7ed with 8-conjugate at 3 weeks of age or at 8 weeks of age. Significant
IgGI levels were elicited by the 8-oligosaccharide-TT-conjugate in mice
ni~ed at 3 weeks old (0.273 ~lg/ml) and at 8 weeks old (0.700 ~g/ml).
5 Tnrlic~tive of a TD response, an adjuvant (e.g., FCA) increased specific IgGI
(1.22 llg/ml). The 8-polysaccharide imillced over-expression of IgG3 and low
IgGl, typical of a polysaccharide TI response. The 8-polysaccharide-TT
conjugate, considered a "TD antigen", in~ ced only low levels of IgGI, with
overexpression of IgG3, characteristic of TI polysaccharide antigens. Also,
1 0 adjuvant in combination with the 8-polysaccharide-TT conjugate did not enhance
IgGI levels, but did increase IgG3 antibody (TI-like response). Some
polysaccharide-conjugates are known to elicit combinations of TI and TD
antibody response profiles (Stein, 1992; Stein, 1994).
1 5 Figure 20 depicts the IgG antibody isotypes elicited by a 8:14 di-hapten-
oligosaccharide-TT conjugate to 8 polysaccharide. Like the 8-mono-hapten
conjugate, this di-hapten conjugate could induce much higher levels of specific
IgGI antibody (a TD response) than a 8-polysaccharide-conjugate or 8-
polysaccharide alone. Overexpression of the IgG3 isotype to polysaccharide
20 immllnngen is shown. Control mice were injected with tetanus toxoid alone.
Results obtained with serotype 14-oligosaccharide conjugates are shown
in Table 21. A 14-oligosaccharide-TT-conjugate prepared by 0.1 M TFA
hydrolysis elicited IgGI, G2a, G2b, and G3 isotypes, the 1 ~g dose was the most
25 immunogenic. Oligosaccharide-TT conjugates prepared using carbohydrate
fractions of separation peaks 7 and 8 of a 0.5 M TFA hydrolysate elicited lower
levels of IgG isotypes. Smaller oligosaccharides (peaks 4 and 5 of the 0.5 M
TFA plepdldlion) in conjugate form elicited low levels of IgG isotypes. The 14-
polysaccharide-TT conjugate elicited relatively high levels of IgGI isotypes.
30 However, serum from mice injected with this polysaccharide conjugate was not
- 21 ~3 733
- 46 -
immllnt)protective (as will be shown in Example 8, Table 24). There appears to
be a required threshold level of IgG antibody isotypes to provide
immllnoprotection to the serotype 14 pathogen. The uncoupled 14
polysaccharide, tetanus toxoid alone, or 0.9 % NaCl negative control serum all
5 displayed low levels of all isotypes, equivalent to normal mouse serum (NMS)
levels.
Figure 21 shows an increased level of IgGl antibody isotype to 14-
polysaccharide elicited by a 8:14 di-hapten-oligosaccharide-conjugate, typical of
1 0 a TD response. The 14-polysaccharide in-luce~l overexpression of IgG3 (TI
response), the 14 oligosaccharide alone was not immllnogenic. Uncoupled
tetanus toxoid was a negative control.
As with individual hllm~n.~, dirrelcllL groups of mice displayed variable
1 5 responsiveness to oligosaccharide- and polysaccharide-conjugates. In certaingroups of mice, variations in the dirrelellL IgG antibody isotype levels were
observed. Figure 22A shows results from a group of "good responser" mice
which produced IgGl to a 14-polysaccharide conjugate (a TD-like response).
Nevertheless, a 14-oligosaccharide-conjugate elicited higher IgGl levels. This
20 conjugate also elicited substantial levels of IgG2b (0.955 ~g/ml =
oligosaccharide-conjugate; 0.139 ~lg/ml = polysaccharide-conjugate). This
response was TD driven as FCA enhanced these IgG2b antibodies, Figure 22B.
(1.509 ~g/ml = oligosaccharide-conjugate; 0.474 ~lg/ml = polysaccharide-
conjugate).
The ability of oligosaccharide-conjugates of the invention, to elicit greater
TD antibody responses than polysaccharide-conjugates was not limited to S.
pneumoniae immllnogens. Oligosaccharide-conjugates of Neisseria meningitidis
Group C elicited greater levels of IgGl isotype antibody (7.01 ~g/ml) than the
30 polysaccharide-conjugate (3.60 llg/ml) or polysaccharide alone (0.162 ,ug/ml).
_ 21 ~3 733
- 47 -
Interestingly, the IgG3 isotype amounts in~ ce~l by the oligosaccharide
conjugates was also more (13.11 llg/ml = oligosaccharide-conjugate; 9.84 ~lg/ml
= polysaccharide-conjugate; 3.81 ~g/ml = polysaccharide alone).
Fx~ml?le 8:
Bactericidal and Opsonization Assays to Measure Tmmllnoprot~tive Antibodies
Elicited by Conjugates
The basic bactericidal and opsonization assays used are as follows:
Bactericidal Assay
1 0 1. Streak a blood agar plate with desired gram negative bacteria procured
from the American Type Culture Collection. Incubate at 37C, overnight.
2. Next day, pick an isolated colony and inoculate it in 1.0 ml of Todd-
Hewitt Broth (THB) + Yeast Extraction (YE) media in a sterile test tube.
Tn~ bate at 37C overnight.
1 5 3. On the following day, measure O.D. of inoclll~te~l bacteria at 420 nm
wavelength. Use THB +YE media as blank.
4. To a sterile flat bottom 96-well plate, add a sterile 2.5 mm glass bead in
each well.
5. To each well, add:
a. 5 ml of bacteria.
b. 10 ml of mouse serum to be tested.
incubate at 37C for 1 hour.
Note: Step # 5 and # 6 are done in triplicate
6. After 1 hour incubation, prepare 1 :20 dilution of exogenous complement
e.g. (Low Tox Rabbit Complement, Cedarlane) sterilely in THB+YE. Add 50
~ll/well. Incubate at 37C for 1 hour.
7. After complement incubation, 50 ml aliquot is plated out on blood agar
plates using a glass spreader.
8. Wrap all agar plates in plastic bags and incubate at 37C for 12 hours.
30 9. Next day, count plaque forming colonies.
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Opsonization Assay
1. Streak a blood agar plate with desired gram negative or positive bacteria
(procured from the American Type Culture Collection). Incubate at 37
5 overnight.
2. Next day, pick an isolated colony and mix it with 1.0 ml of THB+YE
media in sterile test tube. Incubate at 37C overnight.
3. The next day, prepare 100 U/ml of sterile heparin.
4. I.V. inject 100 ml of sterile heparin into tail of each mouse (5 - 10 mice).
10 After 10 minutes, bleed mice retro-orbitally into a sterile tube.
5. Measure O.D. of bacteria at 420 nm wavelength. Use THB+YE media
as blank. (Use spectrophotometer 4040 to measure O.D.)
6. To a sterile flat bottom 96 well plate with sterile 2.5 mm glass bead in
each well, add:
a. 50 ml of hepalil~ized blood.
b. 10 ml of serum
c. 5 ml of bacteria
Do this step in triplicate
7. Wrap plate in tinfoil and incubate at 37C incubator for one hour on a
20 shaker (slow motion.
8. After one hour, a 50 ml aliquot is plated out on blood agar plates using a
glass spreader.
9. Wrap all plates in plastic wrapper and incubate at 37C for 12 hours.
10. Next day, count plaque forming colonies.
Serum from mice immlmi~P~l with a S. pneumoniae type 8
oligosaccharide conjugate was found the be immllnnprotective as measured by
the opsonization assay. Opsonization of S. pneumoniae bacteria mPfli~te~l by
specific anti-capsular antibodies is essential for host defense (Saunders, et al.,
30 1993). This assay is generally considered a reliable indication of
21S3733
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immllnnprotective capability in vivo. Results from assays show that antibodies to
the 8 oligo-conjugate greatly reduce growth of colony forming units of S.
pneumoniae serotype 8 on blood agar plates (Table 22). This reduction was
specific, as colony growth of serotypes 3 and 6B (used as specificity controls)
5 were not inhibited. ~ on with the unconjugated oligosaccharide or
polysaccharide (which is used in the commercially available pneumoniae vaccine)
elicited no protection. Protection elicited with the polysaccharide-conjugate was
much less (39% reduction) than the protection elicited with the oligosaccharide
conjugate (98% reduction). These results demon~tldt~ that our 8 oligo-tetanus
10 toxoid conjugate elicits high levels of immunoprotective antibodies against the
serotype 8 S. pneumoniae pathogen. The level of imml~nnprotective antibody
elicited by poly-conjugates was marginal.
As well, the 8-oligo conjugate could elicit an immllnoprotective antibody
15 response in mice previously ~(lmini~tered the whole polysaccharide alone. Mice
injected with 2 doses of 8-polysaccharide followed by a tertiary oligo-conjugateinjection had immunoprotective antibodies in their serum (70% colony reduction
in opsonization assay). As in previous experiments, mice receiving 3 injections
of polysaccharide elicited no significant amount of protective antibody. Specific
20 oligosaccharide serotypes coupled to a carrier protein may be beneficial as abooster to augment the immunoprotection of high risk groups, non-responsive or
only lllalghlally responsive to the current 23-valent polysaccharide vaccine.
We have performed an immunogenicity study with di-hapten 3 oligo/8
25 oligo-tetanus toxoid conjugates. Oligosaccharides of both serotypes were
prepared by TFA hydrolysis. Mice injected with this multi-hapten conjugate
elicited immunoprotective antibodies to the 3 and 8 serotypes (96 - 99% colony
reduction - Table 23). A 3/8-polysaccharide conjugate elicited little
immunoprotective antibody (10 - 12%). The mono-hapten 3 oligo-tetanus toxoid
30 conjugate used in this study was not prepared with oligosaccharides that had been
21S37~
- so -
determined to have immlmogenic epitopes by inhibition ELISA and was not
capable of eliciting an irnmunoprotective response. The mechanism which
allows the immlm~ system to response to epitopes on the 3 oligosaccharide in thedi-hapten form is, of course, speculative. However, we suggest that the 8
5 oligosaccharides stim~ te clones of cell (i.e. accessory or helper cells) which
can augment the response to the epitopes on the serotype 3 oligosaccharide.
We have discovered that the 8 oligosaccharide structure has adjuvant or
adjuvant "like" activity. The relatively simple repeating unit structure of the 8-
1 0 oligosaccharide (~-glucose (1 ~ 4) ,~-Glucose (1 ~ 4)a-galactose (1 ~ 4) a
gluconic acid) may specifically or non-specifically ctim~ te/activate i~
cells or induce receptors or factors to enhance a humoral/cellular response to
non-immlmogenic or weakly immlm~genic polysaccharides/oligosaccharides.
Serotype 8 oligosaccharides has adjuvant activity in conjugate form or as an
1 5 ~(lmixtllre to the vaccine formulation.
Opsonization results of a 14-oligosaccharide-TT conjugate (0.1 M TFA
preparation - Table 24) show good bacterial colony reduction of the 14 serotype
(76%). The 14-oligo-TT 0.5 M TFA plepal~tion elicited less immllnoprotective
20 antibody (54% reduction). The serums from the polysaccharide-TT conjugate,
the polysaccharide alone and the tetanus toxoid injected mice showed greatly
reduced inhibition capacity (18, 2 and 15% respectively). Serum from control
mice (0.9 NaCL injected and NMS) showed no reductive capacity.
Di-hapten-oligosaccharide conjugates also elicited antibody with opsonic
activity. A serum to a 8:14-oligo-TT conjugate reduced serotype 14 colony
forming units by 65% (Table 25). This di-hapten conjugate was as immlmogenic
as the mono-hapten 14-conjugate (reduction of CFU = 68%). Serum from mice
immllni~ed with the polysaccharide-conjugate marginally reduced CFU's by
30 37%.
- Sl - 21 $3 733
Example 9:
Circumvention of Carrier Suppression and Reduction of Antigenic Competition
~educed responses due to antigenic competition when multiple antigens
are injected has been reported in the literature under some conditions. Results
obtained from i.,~"~ni~ ion schedules A and D (Table 26) will be used to
determine if the response to each component of our multi-hapten conjugate is
equal to the response elicited by the single mono-hapten conjugates.
The unit mass of carbohydrate antigen of our mono- and multi-hapten
conjugates will be equivalent (i.e., l :2 CHO:protein ratio for EDC conjugates).The design of our multi-hapten conjugates using reduced antigen load will
",il~i",i,e the potential for developing antigenic colllpc;Lilion.
1 5
Schedules B and E will determine if a plilllaly injection with the
conjugate is sufficient to educate the immlm~ system to elicit a T dependent
response when boosted with uncoupled polysaccharide(s).
Schedules C and F will establish the capability of our conjugates to
enhance immllnoprotective antibody responses in mice previously primed with
polysaccharide(s) alone. If so, a multi-hapten pneumoniae vaccine coll~ g
oligosaccharides of 3 to 4 serotypes may be very useful to augment the response
to Pneumovax~ 23 in high risk patients.
Groups of mice will be injected by 3 doses (1, 2, 3) of tetanus toxoid
(titers to tetanus toxoid to be confirmed by ELISA) followed by 3 injections of
various S. pneumoniae oligo or poly-TT conjugates as in G (Table 26).
In all studies, conjugates will be ~tlmini.ctered orally and by subcutaneous
injection.
-
- 52 - 21 ~3 73~
The conjugates of the present invention will stimnlAte immnn~ responses
in infants, in children with i~ llAIIlle imml-n~ ~y~Lell~s and in the
immlmosuppressed. As models for these situations, we will d~le.ll-il~e the
5 immnn~po~ g efficacy of our conjugates in young mice, in SCID and nude
mice. As described above, these mice will also be pre-sensitized with tetanus
toxoid prior to multi-conjugate inoculation to study the carrier ~upplession
phenomenon.
Modification of the above-described modes of carrying out the various
embodiments of this invention will be ~al~llL to those skilled in the art
following the te~c-hing~ of this invention as set forth herein. The examples
described above are not limiting, but are merely exemplary of this invention, the
scope of which is defined by the following claims.
